Oro solution

ORO staining was conducted to identify the adipogenic committed cells. Briefly, the BMSCs were fixed using 4% PFA solution, dyed with ORO solution (Cyagen Biosciences, China) for 30 min, and then observed through an inverted light microscopy (Nikon, Japan). Finally, the representative images were acquired and the number of adipocytes was obtained.

BMSCs were plated in 48-well plates at a density of 3.0 × 10⁵ cells mL^-1 for osteogenic induction and 6.0 × 10⁵ cells mL^-1 for adipogenic induction. Twenty-four hours later, the culture medium was replaced with fresh osteogenic or adipogenic medium (Cyagen Biosciences, Guangzhou, China) supplemented with B-EVs (100 μg mL^-1 at the protein level) from different groups (WT-B-EVs, AD-B-EVs, or an equal volume of vehicle). The medium was replaced with fresh medium containing the corresponding supplements every 48 h for osteogenic induction, or 72 h for adipogenic induction. After differentiation for 3 days, the total RNA of the differentiated BMSCs was extracted and the expression of pro-osteogenic/anti-adipogenic transcription genes related to osteogenesis or adipogenesis was assessed by qRT-PCR. After differentiation for 8 days, the cells were stained with ARS solution (Cyagen, Suzhou, China) to evaluate matrix mineralization. After differentiation for 12 days, the cells were stained with ORO solution (Cyagen, Suzhou, China) to detect lipid droplet formation. The percentages of ARS- and ORO-positive areas were analyzed from five random visual fields for each biological replicate and three biological replicates for each group. All in vitro induction experiments were repeated 3 times.